title: Antipeptide monoclonal antibodies to defined fibrinogen Aalpha chain regions: anti-Aalpha 487-498, a structural probe for fibrinogenolysis.
abstract: The fibrinogen alphaC domain (Aalpha 220-610) is one of the earliest targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the alphaC domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its Aalpha chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb F-104 was raised against a multiple antigenic peptide derivative modelled after the hydrophilic 12-residue sequence corresponding to Aalpha 487-498 within the alphaC domain. A sensitive solution phase competitive enzyme-linked immunosorbent assay (ELISA) was developed for MoAb F-104 that can be applied for the direct measurement of intact fibrinogen (purified or plasma; ED50% approximately 5 pmol Aalpha chain equivalents/mL), with negligible cross-reactive interference from peptide cleavage products released by plasmin from the COOH-terminal end of the Aalpha chain (<3%). Immunoblotting and ELISA studies to characterize the fate of the F-104 epitope during fibrinogenolysis in vitro indicated a rapid loss of fibrinogen-associated immunoreactivity that reflected the heterogeneity of plasmin cleavage sites within the alphaC domain; cleavage at the 493-494 arg-his bond destroyed the F-104 epitope, while cleavage at other sites released it in an altered, inaccessible, conformation within the structure of 35- to 40-kD and 17.5- to 18-kD Aalpha chain degradation products. Application of the F-104 ELISA to monitor the course of Aalpha chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that the loss of fibrinogen-associated F-104 immunoreactivity was a very early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a reagent for evaluating the creation of an effective lytic state early during therapy, information that could help determine the need for further clinical intervention. Thus, these studies illustrate a rational, targeted, approach towards the development of a novel antifibrinogen MoAb whose application as a structural probe for the region Aalpha 487-498 in vitro and in vivo can provide new insights into the various molecular forms of fibrinogen that circulate under physiologic conditions and in disease.
authors.affiliation: Department of Medicine, College of Physicians & Surgeons of Columbia University, New York, NY 10032, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Sobel
authors.forename: I
authors.initials: I
authors.lastname: Trakht
authors.forename: N
authors.initials: N
authors.lastname: Pileggi
authors.forename: H
authors.initials: H
authors.lastname: Qi Wu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000276:immunology; D005340:Fibrinogen / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism*; D005342:Fibrinolysis; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D009203:Myocardial Infarction / Q000188:drug therapy; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D013045:Species Specificity; D015912:Thrombolytic Therapy
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D005340:Fibrinogen; D005341:Fibrinolysin